Defects in the mitochondrion, the energy-producing unit of the cell, lead to a wide range neural and muscular diseases caused by decreased energy production, free radical damage, and perturbations to apoptotic pathways. The protein import pathways of the mitochondrion mediate the import and assembly of proteins from the cytosol. The X-linked disease Mohr-Tranebjaerg syndrome or deafness-dystonia syndrome is caused by a specific defect in the import of inner membrane proteins. The goal of this proposal is to investigate the mechanism of protein import into the mitochondrion in the experimental model, the budding yeast Saccharomyces cerevisiae. Components of this import pathway include the soluble Tim8p-Tim13p and Tim9p-Tim10p complexes in the intermembrane space and the TIM22 complex (Tim12p, Tim18p, Tim22p, and Tim54p) in the inner membrane. The objective of this research is to define the molecule mechanisms of protein import with a combined biochemical, biophysical, and genetic approach. Specifically, the mechanism by which the Tim8p-Tim13p and Tim9p-Tim10p complexes are assembled and subsequently escort the substrates to the inner membrane will be elucidated. In addition, a chemical-genetic approach will be utilized to identify small molecule effectors that may modulate the import pathway, with the long-term goal of developing therapeutics for deafness-dystonia syndrome and other mitochondrial disorders. Moreover, the link between mitochondrial protein assembly and degradation pathways will be investigated in our yeast models. The proposed project will expand fundamental knowledge about the mechanism of protein insertion into the mitochondrial inner membrane, extending present studies that have focused generally on the mechanism by which proteins reach soluble compartments of the mitochondrion. This research will impact public health because these mechanistic studies will provide insight into how defects in mitochondrial biogenesis lead to diseases such as deafness-dystonia syndrome, Friedreich's ataxia, and Parkinson's and Alzheimer's disease, which are caused by mitochondrial dysfunction. The goal of this research is to use our yeast models in a chemical-genetic approach to identify small molecule effectors, which in the long-term may lay the groundwork for developing new tools to understand how mitochondrial defects lead to human diseases and new therapeutic approaches to develop drugs that will modulate mitochondrial function.